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Abstract:

Said preparations can be administered topically to the intact eye
surface, and are useful in the prevention and treatment of
neurodegenerative disorders of the retina, optic nerve, lateral
geniculate body and visual cortex, in order to prevent reduction of
visual capacity and restore the normal visual function.

Claims:

1. Ophthalmic preparation in the form of eyedrops comprising a
Brain-Derived Neurotrophic Factor (BDNF) in a concentration of at least
15 μg/μl.

13. A method of preventing and/or treating neurodegenerative diseases of
the retina, optic nerve and lateral geniculate body, said method
comprising: administering an effective amount of an ophthalmic
preparation in the form of eyedrops comprising BDNF to a subject in need
thereof.

Description:

TECHNICAL FIELD OF INVENTION

[0001] The present invention relates to ophthalmic preparations in the
form of eyedrops, which contain the brain-derived neurotrophic factor
(BDNF) and a viscosity-controlling agent, preferably a galactoxyloglucan
extracted from tamarind seeds, also known as TS-polysaccharide or TSP.

[0003] Neurotrophins are proteins synthesised by the nerve cells which
control the survival and normal trophism of various cells present in the
nervous system.

[0004] The best known is nerve growth factor (NGF), discovered by R.
Levi-Montalcini and S. Cohen in the mid-20th century.

[0005] Other factors, whose protein structure presents structural
similarities with that of NGF, were discovered later; consequently, we
now consider a class of NGF factors (neurotrophins) to which BDNF, NT-3,
NT-4/5 and NT-6 belong as well as NGF (the first three are mainly
expressed in the nervous system of mammals, while NT-6 is a new member of
neurotrophins identified in teleost fish and absent in mammalian brain).

[0006] Neurotrophic factors, including neurotrophins, are released by
nerve cells which synthesise them, and bind to specific receptors on the
membrane.

[0007] Despite their structural similarities, the various neurotrophins
act through different receptors, and consequently through different
action mechanisms.

[0008] Binding of a neurotrophic factor to its specific receptor (TrkA for
NGF; TrkB for BDNF, and partly for NT-4; TrkC for NT-3) generates a
cascade of events, which elicit a specific response by the nerve cell.

[0009] Different neurotrophin receptors are expressed in different areas
and, within the same area, in different cells, activating specific
intracellular signal transduction pathways. It therefore follows
logically that not all areas or nerve cells can respond to each of the
four neurotrophins; the limiting factor is the cell distribution of the
specific receptor for a given neurotrophin.

[0010] The distribution of retinal cells able to synthesise and release
NGF, the archetype of the neurotrophins, and the distribution of retinal
cells that express the NGF receptor (TrkA), appears very limited, and is
restricted in practice to a sub-group of ganglion cells and astrocytic
glial cells (Garcia et al., 2003).

[0012] As reported by Lambiase et al., these preparations increase the
retinal levels of NGF; however, it can be demonstrated that NGF is unable
to perform a neuroprotective effect on the retina.

[0013] This agrees with the findings recently reported by Shi et al.
(2007). NGF can bind to two types of receptor in the retina, TrkA and
P75, which exert opposite effects on the trophism and survival of the
nerve cells. When exogenous NGF reaches the retina, it may therefore
induce two opposing effects on the retinal cells, which tend to cancel
each other out.

[0016] WO 97/45135 relates to stable pharmaceutical compositions of BDNF
in the form of an aqueous solution or lyophilisate. In that document,
especially in the section devoted to the prior art, BDNF is mentioned as
being useful in the treatment of various disorders, including retinitis
pigmentosa. The only form of administration expressly mentioned is
injectable preparations.

[0017] JP 2003048851 relates to ophthalmic formulations based on BDNF, to
be administered in the form of drops on the conjunctiva. The formulations
disclosed contain various viscosity-controlling agents, described as
being equally effective in carrying BDNF to the retina.

[0018] The evidence of activity reported in said document is unconvincing,
because the concentration range indicated for BDNF is very wide: 0.001-1
weight/volume %, corresponding to a concentration range of between
1×10-2 and 10 μg/μl [claim 3, ambit of patent; according
to the detailed description of the invention [0006], paragraph 3, but in
the example reported, the concentration used is=0.004% (weight/volume%),
corresponding to 4×10-2 μg/μl, ie. much lower than the
effective concentrations able to increase the retinal levels of BDNF and
prevent the retinal alterations induced by lengthy exposure to light,
which are equal to or greater than 15 μg/μl, in the 15-200
μg/μl range, in agreement with the present invention. It should be
noted that in JP 2003048851 the application was repeated three times a
day (10 μl/application, 0.004% weight/volume) for 5 days, equal to a
dose of 1.2 μg/day and a total dose of 6 μg. Even if the daily dose
and the total dose are taken into account, they are too low to exert
neuroprotective effects; in fact, in agreement with the present
invention, a minimum total dose of 150 μg had to be administered
topically to obtain neuroprotective effects in the retina subjected to
light damage. New data obtained with another experimental model, namely a
mouse that develops glaucoma, confirm that of the three BDNF
concentrations used (1, 5 and 15 μg/μl), only the highest (15
μg/μl) is effective.

[0019] Moreover, JP 2003048851 refers to a retina-protecting effect
verified by histological techniques (staining of retina sections with
haematoxylin-eosin) designed to measure retinal thickness, but not
accompanied by a demonstration of restoration of the retinal function
measured by a flash electroretinogram recording, as reported in the
present invention. It is well known that in order to demonstrate the
neuroprotective efficacy at retinal level of any treatment, results
obtained with histological/morphological techniques only are
insufficient; evidence of restoration of the retinal functions is also
required. It can therefore be concluded that in JP 2003048851, the
ophthalmic compositions of BDNF in the concentrations reported in the
examples, and more generally in the preferred range, administered
externally, are unable to pass from the eye surface to the internal
tissue in quantities sufficient to exert a neuroprotective effect able to
restore the retinal function.

[0020] WO 2006/046584 relates to sustained-release compositions containing
HGF, BDNF or PEDF, impregnated with a cross-linked gelatin hydrogel,
useful in treating disorders involving lesions of the visual cells, such
as retinitis pigmentosa degeneration. In the specific examples, the
compositions take the form of microspheres containing doses of BDNF of
between 0.001-1000 μg, and can be administered by intraocular
injection or subretinal implant.

[0021] EP 0 958 831 describes ophthalmic compositions containing a
neurotrophic factor selected from a group of factors including BDNF. Said
compositions can be applied externally, for example in the form of
ophthalmic ointments or solutions, or can be formulated as contact
lenses.

[0022] The concentration of neurotrophic factor disclosed in EP0958831
ranges from 0.0001 to 0.5% (weight/volume), ie. from 1×10-3 to
5 μg/l. The concentration ranges reported are therefore very wide.
EP0958831 is highly generic, because it relates to various neurotrophic
factors, including BDNF, which would be equally effective in the
concentration range. It is known that neurotrophic factors are not
equally effective in the same concentration range, due to the different
densities and distribution of the receptors that determine their
biological effects in the different brain areas and individual nerve
cells.

[0023] Moreover, EP 958831 is extremely vague and unclear about the range
of effective BDNF concentrations for topical use: on p. 3 line 44 (see
paragraphs 0022 and 0033, claims 19 and 20) two concentration ranges are
given, which do not match (range A maximum, between 0.0001 and 0.5%
(W/V), equal to a concentration range of between 10-3 and 5
μg/μl, and range B, between 10-3 and 2×105 μg/l;
the two concentration ranges clearly do not correspond. According to the
present invention, however, the effective concentrations of BDNF are
equal to/greater than 15 μg/μl (range 15-200 μg/μl, ie.
higher than range A reported in EP 958831, namely the maximum
concentration range. The examples in EP 958831 relate to ophthalmic
compositions characterised by a BDNF concentration of 0.02, 0.04 and 10
μg/l, ie. concentrations which are far lower (1×106 times
lower) than the lowest concentration which, according to the present
invention, has proved effective in raising the BDNF levels in the retina
and preventing both light damage and glaucoma damage, namely 15
μg/μl.

[0024] It can therefore be concluded that ophthalmic compositions of BDNF
at the concentrations reported in EP 958831, administered externally,
cannot pass from the eye surface to the internal tissues in quantities
sufficient to increase the retinal levels of BDNF, and consequently to
perform a therapeutic effect.

[0025] NT-4, the other neurotrophin which binds to TrkB, is expressed at a
low level in the retina, and only acts on a sub-group of amacrine cells,
ie. those which synthesise dopamine (Calamusa et al., 2007).

[0026] The absence of BDNF or its receptor causes serious alterations in
the retinal function; for example, mice that lack the TrkB receptor
(knock-out mice) are characterised by complete loss of the retinal
response to light (total absence of b-wave in the flash
electroretinogram; Rohrer et al.,1999).

[0027] LaVail's group has demonstrated that intraocular injections of
BDNF, but not NGF, effectively prevent morphological degeneration of the
photoreceptors induced by light damage.

[0028] Intraocular injections of BDNF together with other neurotrophic
factors have reduced the damage to the retinal ganglion cells which
results from lesions of the optic nerve, although it is not yet clear
whether BDNF is able to perform neuroprotective effects alone, ie.
independently of other neurotrophic factors (Watanabe et al., 2003; Yata
et al., 2007).

[0029] Other neurotrophic factors such as FGF2 have proved equally
effective in preventing morphological alterations caused by light damage,
but unlike BDNF, their administration has the undesirable effect of
activating factors involved in the inflammatory response (LaVail et al.,
1987).

[0031] These results indicate that for neuroprotective purposes in retinal
disorder models, it is not sufficient to evaluate the morphological
effects of neuroactive molecules; above all, it is essential to assess
whether those molecules exert protective effects on the retinal function,
and ensure that they do not impair the response of the retinal cells to
visual stimuli. There is currently a need to identify new BDNF
preparations which can be administered by non-invasive techniques to
convey BDNF to the retina, avoiding highly invasive administration
techniques such as intraocular, subretinal or retrobulbar injections,
which are unsuitable for long-term chronic treatment due to the risk of
causing perforation of the eyeball, infections or bleeding, for example.

[0032] The present patent proposes topical conjunctival applications in
various formulations containing BDNF in a concentration range of between
15 and 200 μg/μl, with a total BDNF dose of between 50 and 4000
μg per administration, according to the size of the eye to be treated,
which will depend on the animal species concerned, including humans.

[0033] The formulation will preferably contain a viscosity-controlling
agent. Said viscosity-controlling agent is preferably a galactoxyloglucan
extracted from tamarind seeds (TS-polysaccharide or TSP) having a
molecular weight of between 500000 and 800000 Da and the following
structural formula:

##STR00001##

[0034] We demonstrate that said preparation, at the concentrations and
dose per administration indicated, significantly increases the retinal
BDNF levels and prevents i) retinal alterations induced by lengthy
exposure to light, and ii) retinal alterations in glaucoma.

[0035] It has previously been demonstrated (Uccello-Barretta G et al.,
2008; Ghelardi E et al., 2004; Burgalassi S et al., 2000; Ghelardi E et
al. 2000) that TSP is able to carry pharmacologically active molecules
for topical treatment of the eye surface. By increasing the retention
times of the formulation on the eye surface, increased absorption of the
active molecules has been observed. This property of TSP has been
described in combination with antibiotics (rufloxacin, gentamicin and
ofloxacin), antihistamines (ketotifen) and antihypertensives (timolol),
all of which are small molecules.

[0036] In the case of pharmacological preparations containing recombinant
proteins, such as BDNF, the active ingredient is a protein, a molecule
with high molecular weight subject to post-translational modifications
and adaptation of its spatial bending until it reaches the active
three-dimensional configuration. The biological activity of proteins is
closely dependent on their three-dimensional configuration, because
interactions with the specific receptors and enzymes that recognise them,
and consequently, the ability to intervene in the biochemical processes
of the target cell, depend on it. The protein configuration can be
considerably modified by the environment in which a recombinant protein
is to be found. Formulations containing recombinant proteins must
therefore ensure that the protein is maintained in solution in its active
configuration, and guarantee its stability.

[0037] The present invention demonstrates that TSP guarantees the
stability of BDNF in the formulation, increases its ocular absorption due
to the lengthy residence time of the formulation on the surface, and
above all, maintains BDNF in its biologically active configuration.

SUMMARY OF THE INVENTION

[0038] It has now been discovered that ophthalmic formulations containing

[0039] BDNF in concentrations of at least 15 μg/μl prevent retinal
alterations induced by lengthy exposure to light, and those associated
with increased intraocular pressure in a glaucoma model.

[0040] The invention therefore relates to ophthalmic preparations in the
form of eyedrops containing brain-derived neurotrophic factor (BDNF).
According to a preferred aspect thereof, the compositions to which the
invention relates contain galactoxyloglucan extracted from tamarind
seeds, known as TSP.

[0041] The invention also relates to the use of BDNF to prepare a
medicament in the form of eyedrops for the prevention and/or treatment of
neurodegenerative disorders of the retina, optic nerve and lateral
geniculate body.

[0042] The present invention also relates to an ophthalmic preparation in
the form of eyedrops containing BDNF for use in the prevention and/or
treatment of neurodegenerative disorders of the retina, optic nerve and
lateral geniculate body.

LIST OF FIGURES

[0043] FIG. 1--Determination of BDNF levels in the retina (A), optic nerve
(B) and vitreous humour (C) following topical application of BDNF in
saline solution. The BDNF levels are shown on the y-axis, and were
measured in the eye treated with BDNF and the control eye treated with
saline solution; * indicates the significance of the differences.

[0044] FIG. 2--Determination of BDNF levels in the retina (A), optic nerve
(B) and vitreous humour (C) following topical application of BDNF in
solution with sodium carboxymethylcellulose. For conventions and symbols,
see FIG. 1.

[0045] FIG. 3--Determination of BDNF levels in the retina (A), optic nerve
(B) and vitreous humour (C) following topical application of BDNF in
solution with TSP. For conventions and symbols, see FIG. 1.

[0046] FIG. 4--Comparative efficacy of TSP, saline solution (NaCl) and
sodium carboxymethylcellulose (CMC) in carrying BDNF and increasing its
retinal concentrations (pg/mg of protein, see x-axis). The retinal levels
of BDNF after topical treatment with BDNF in TSP (*) significantly
increased, compared with the BDNF level after topical treatment with BDNF
in saline solution and sodium carboxymethylcellulose. The data are
derived from panel A in FIGS. 1, 2 and 3.

[0047] FIG. 5--Kinetics of BDNF levels in the retina, optic nerve and
vitreous humour following topical application of BDNF in solution with
TSP. The BDNF levels remain significantly high in the first 6 hours after
treatment (*).

[0048] FIG. 6--Topical application of BDNF in TSP reduces light-damage
induced alterations of the retinal flash response (flash ERG). The
amplitude (μV, see y-axis) of the b-wave of the electroretinogram
evoked by flashes of different luminances (cd/m2, see x-axis) and
recorded by the eye treated with BDNF (black symbols) or the control eye
treated with TSP only (light-damaged control, white symbols) was
measured; * indicates that the differences are significant.

[0049] FIG. 7--Topical treatment with BDNF in TSP increases the number of
photoreceptors surviving light damage. The photoreceptors are labelled
with propidium iodide in cross-sections of retina. Regardless of the
method used (photoreceptor cell body row count (FIG. 7B) or measurement
of thickness of the outer nuclear layer (ONL) (FIG. 7C)), the
photoreceptors present in the central and peripheral retina are
significantly (*) more numerous in the eye treated with BDNF than the eye
treated with the carrier (control).

[0050] FIG. 8--Topical application of BDNF in saline solution (NaCl)
reduces light-damage induced impairment in the retinal response to light.
For an explanation of conventions and symbols, see FIG. 6.

[0052] FIG. 10--Topical application of BDNF in solution with sodium
carboxymethylcellulose, and impaired response to light induced by light
damage. For conventions and symbols, see FIG. 6.

[0053] FIG. 11--Effects of topical treatment with BDNF in solution with
sodium carboxymethylcellulose on photoreceptor survival after light
damage to the eye treated with BDNF and the eye treated with the carrier
(control). For conventions and symbols, see FIG. 7.

[0054] FIG. 12--Increase in intraocular pressure (TOP, mmHg) in an
experimental murine glaucoma model, DBA/2J mouse v. normal mouse
(C57b1/6J). The increase in TOP in DBA/2J is significant (*) as from the
age of 61/2 months.

[0055] FIG. 13--The response of the retina to visual patterns (pattern
ERG, P-ERG; stimulus consisting of a spatial frequency=0.2 C/deg,
contrast 90%) was recorded in normal mice (C57b1/6J, dark bar) and
glaucoma-developing mice (DBA/2J); the amplitudes of the responses of
P-ERG (μV, see y-axis) were measured by stimulating the eye treated
for two weeks with different concentrations of BDNF (1, 5 and 15
μg/μl ) and by stimulating the eye treated with the carrier,
considered as control eye (CTRL); * indicates the significance of the
differences. P-ERG was recorded at the age of 7 months in the DBA/2J
mouse, ie. after the increase in intraocular pressure (TOP).

[0056] FIG. 14--Retinal ganglion cells (whole-mount preparation) of
glaucoma-developing DBA/2J mouse (aged 7 months), labelled with a
fluorescent antibody that binds to a transcription factor (Brn3b). A. The
left-hand column shows the effects of two weeks' topical treatment with
BDNF in TSP in the central retina (top row) and peripheral retina (bottom
row). Following treatment with BDNF (left-hand column), the labelled
cells were more numerous than in the retina of the control eye (CTRL),
which was treated with the carrier only (right-hand column). B.
Quantitation of effects of topical treatment with BDNF on ganglion cells
(density measured in cells/mm2, shown on the y-axis) labelled with
Brn3b in the glaucoma-developing mouse (DBA/2J; eye treated with BDNF;
eye treated with carrier, CTRL) and in the normal mouse (C57b1/6J).

DETAILED DESCRIPTION OF THE INVENTION

[0057] It has surprisingly been found that by administering exogenous
brain-derived neurotrophic factor (BDNF), applied topically to the intact
eye surface, in particular in the conjunctival sac, BDNF performs a
neuroprotective effect on the retinal cells at both functional and
morphological levels, thus allowing the prevention and/or treatment of
neurodegenerative retinal disorders.

[0058] BDNF has demonstrated neuroprotective efficacy not only towards the
photoreceptors, but also towards the ganglion cells, ie. the cells of (i)
innermost layer of the retina, which send their fibres to the visual
centres, (ii) the optic nerve fibres, and (iii) the extra-retinal visual
centres, such as the lateral geniculate body.

[0060] Said ophthalmic preparation contains BDNF in a concentration which
can range between the lower limit of 15 μg/μl and 200 μg/μl,
preferably between 20 and 100 μg/μl, and even more preferably
between 30 and 50 μg/μl. The total bioavailable dose can be between
50 and 4000 μg per administration, according to the volume of the
ophthalmic formulation administered and the species to which the treated
eye belongs, including humans.

[0061] BDNF can be administered alone or in combination with other active
ingredients, such as β-blockers, prostaglandins and carbonic
anhydrase inhibitors.

[0062] The preparation is made in the form of eyedrops, and can be a
solution, a suspension, a gel or an ophthalmological ointment with the
active ingredient BDNF, or active ingredients, in a pharmaceutically
acceptable carrier compatible with the active ingredient and tolerated by
the eyes.

[0063] The pharmaceutically acceptable carrier can be a saline solution,
preferably containing 0.9% of sodium chloride.

[0064] It has also been found that the absorption levels of improved BDNF
can be increased if at least one pharmaceutically acceptable carrier is
used in the preparation, preferably a galactoxyloglucan extracted from
tamarind seeds (TSP) which, due to its viscosity, allows a longer BDNF
residence time on the eye surface than administration in saline solution,
which is washed away from the conjunctiva more quickly.

[0065] The TSP concentration can vary, preferably from 0.05 to 2%
(weight/volume--w/v), and even more preferably from 0.25 to 0.5% (w/v).

[0066] TPS is transparent, viscoelastic and sterile, and is used for
corneal protection. TSP also forms a long-lasting film on the eye
surface, which lubricates and moistens the cornea and conjunctiva.

[0067] According to a further preferred aspect, the viscosified solution
contains hyaluronic acid, and even more preferably, hyaluronic acid
combined with TSP.

[0068] The hyaluronic acid concentration can vary, preferably from 0.05%
to 0.8%% (w/v), and even more preferably from 0.2 to 0.4% (w/v).

[0069] According to a preferred embodiment, the preparation can include
BDNF in the concentration of 15 μg/μl in a saline solution
containing 0.9% NaCl.

[0070] According to a further preferred embodiment, the preparation can
contain BDNF in the concentration of 15 μg/μl in saline solution
with TSP, preferably an 0.25% solution.

[0071] The eyedrop preparation can be administered topically directly to
the intact eye surface, ie. in a non-invasive way, avoiding the use of
invasive methods such as intraocular, subretinal and retrobulbar
injections. In particular, the preparation can be administered into the
conjunctival sac. The preparation can also be formulated as an eyepatch
or in contact lenses.

[0072] The retina is a partly separate part of the central nervous system;
various types of barrier exist, including the blood-retinal barrier,
which prevents the non-specific diffusion of compounds such as large
molecules to the retina. The intraocular penetration of pharmacologically
active compounds applied topically is regulated by barriers located in
the cornea and the conjunctiva, by systemic absorption and by metabolic
breakdown effected by the enzymes present in those tissues. Once
instilled, the pharmacologically active compounds must cross a complex
system of blood barriers, including the blood-retinal barrier, to
penetrate the underlying tissues as far as the retina.

[0073] Moreover, the retina, through the ganglion cells, from which the
optic nerve fibres originate, is connected via the optic nerve to visual
centres such as the dorsal part of the lateral geniculate body (dLGN).

[0074] As demonstrated in the experimental part, BDNF, when administered
topically according to the invention, can be conveyed to the retina,
inducing an increase in its retinal concentration to levels which perform
neuroprotective effects from both the functional and the morphological
standpoint.

[0075] It has also surprisingly been found, as demonstrated by
experimental evidence, that the ganglion cells allow anterograde
transport of BDNF, allowing BDNF to prevent and treat degeneration not
only of the ganglion cells but also of the optic nerve fibres, and
extension of the disorder to the extra-retinal visual centres, such as
the lateral geniculate body.

[0076] The present invention also relates to the use of BDNF to prepare an
ophthalmic medicament in the form of eyedrops for topical administration
to the intact eye surface for the prevention and/or treatment of
neurodegenerative disorders of the retina, optic nerve and lateral
geniculate body, in particular degenerative retinopathies (such as
retinitis pigmentosa and glaucoma), age-related retinopathies (such as
age-related macular degeneration), vascular and proliferative disorders
of the retina, detachment of the retina and retinopathy of prematurity
(ROP) and diabetic retinopathy, which lead to blindness. The preparations
according to the invention are useful for the prevention and/or treatment
of neurodegenerative disorders of the retina, optic nerve and lateral
geniculate body, especially, for example, retinitis pigmentosa and
glaucoma (including congenital glaucoma, infantile glaucoma, juvenile
glaucoma, adult glaucoma, primary open-angle glaucoma, primary
angle-closure glaucoma, acute glaucoma, iatrogenic glaucoma and secondary
glaucoma).

[0077] Glaucoma is one of a series of progressive disorders affecting the
eye which, if not suitably treated, lead to blindness due to loss of
ganglion cells and progressive atrophy of the optic nerve fibres.

[0078] Glaucoma is characterised by an increase in intraocular pressure
(TOP) which can damage the ganglion cells and the optic nerve fibres
either directly (mechanically) or indirectly by inducing ischaemia of the
retinal vessels that supply the inner retina. At the progressive stage,
as well as the retina, glaucoma can affect the visual centres, such as
the lateral geniculate body, until the visual cortex is eventually
involved.

[0079] It has also been found that treatment with BDNF in effective
concentrations not only prevents and reduces photoreceptor degeneration
induced by prolonged exposure to light (light damage) and preserves the
retinal response to light; moreover, the use of an experimental glaucoma
model demonstrates that topical application of BDNF prevents the
degeneration of the retinal ganglion cells which results from a rise in
intraocular pressure (IOP) in an animal glaucoma model; in both animal
models, BDNF did not alter the retinal response to visual stimuli.

Determination of BDNF Levels in Vitreous Humour, Retina and Optic Nerve
After 6 Hours' Topical Treatment of the Eye with BDNF-Based Preparations

[0090] Preparations 1, 2 and 3 Containing BDNF, as Described Above, were
Used:

[0091] The test was conducted on albino rats (Wistar rats, Harlan, Italy);
BDNF in saline solution with sodium carboxymethylcellulose, or in saline
solution with TSP, was applied topically, being instilled into the
conjunctival sac of one eye, while the other eye, used as control, was
treated with the solution ("placebo") used to include and carry BDNF.

[0092] Determination of BDNF Levels in the Retina, Vitreous Humour and
Optic Nerve

[0093] The animals were killed 6 hours after the application, after
induction of deep anaesthesia with an intraperitoneal urethane injection
(20%). The eye was then removed, and the BDNF level measured in the
vitreous humour, retinal homogenate and homogenate of optic nerve of both
the eye treated with BDNF and the other eye treated with the carrier
solution only (control eye). The measurements were conducted by
immunoassay (ELISA; BDNF Emax immunoassay system, Promega, Madison, Wis.,
USA). The quantity of BDNF in the optic nerve was also determined, to
establish whether BDNF introduced from the exterior by topical
application was taken up and conveyed by the retinal cells, in particular
by the retinal ganglion cells which, with their fibres, form the optic
nerve.

[0094] The results shown in the chart in FIG. 1 were obtained with a
topical application of BDNF in saline solution (0.9% NaCl), and are
expressed as mean BDNF concentration values in the retina (A), optic
nerve (B) and vitreous humour (C), expressed as pg/mg of protein.

[0095] The statistical analysis was conducted with Student's t-test,
comparing the eye treated with BDNF with the control eye: in all cases,
the differences between the treated eye and the control eye were
statistically significant (*, p<0.05).

[0096] The results shown in the charts in FIG. 2 relate to a topical
application of BDNF in solution with sodium carboxymethylcellulose
(0.2%), while the results shown in the charts in FIG. 3 were obtained
with a topical application of BDNF in solution with TSP (0.25%); in both
cases, the statistical analysis was conducted with Student's t-test,
comparing the eye treated with BDNF with the control eye. In all cases,
the differences between the treated eye and the control eye were
statistically significant (*, p<0.05).

[0097] FIG. 4 shows the comparative levels of BDNF in the retina for each
type of solution/carrier used; this analysis makes it easier to compare
the efficacy of the different solutions/carriers at the same BDNF
concentration (10 μl of solution containing 150 μg of BDNF).
Topical treatment with BDNF in TSP produced significantly higher retinal
levels of BDNF than the other two formulations used, ie. BDNF in saline
solution and BDNF in solution with sodium carboxymethylcellulose
(Student's t-test *, p<0.05). Topical treatment with BDNF in solution
with sodium carboxymethylcellulose proved to be the least effective in
increasing the retinal BDNF level.

2.2 Example

Determination of BDNF Levels in the Retina, Vitreous Humour and Optic
Nerve at Different Times After Topical Treatment of the Eye with BDNF in
TSP

[0098] The extent to which BDNF remained high in the retina, vitreous
humour and optic nerve after a single topical application was studied.
This study was conducted with the carrier containing TSP, which proved
the most effective in facilitating the transscleral passage of BDNF to
the retina, optic nerve and vitreous humour. The kinetics of the BDNF
levels in the retina, optic nerve and vitreous humour after topical
treatment of the eye were then studied, using TSP. N=5 eyes were treated
in each test group. The BDNF concentration in the retina, optic nerve and
vitreous humour was measured at different intervals of time after
application of an 0.25% solution of TSP containing BDNF (10 μl of a
solution containing 150 μg of BDNF). The control eye was treated only
with the carrier solution containing 0.25% TSP. This experiment was
performed to establish the time trend of the BDNF levels after a single
topical application. The charts show the mean BDNF values (y-axis; pg/ml)
in the retina, optic nerve and vitreous humour 6, 12 and 24 hours after
the application. FIG. 5 shows that the BDNF level in the retina remains
statistically high, returning to baseline levels in 12-24 hours. The
statistical analysis was conducted with Student's t-test: in A *,
p<0.01 compared with the control eye. The results of this experiment
suggest that in long-term treatment with BDNF, carried in artificial
tears, in particular based on TSP, one topical application every 12 hours
is sufficient to maintain high BDNF levels in the retina.

[0099] To establish the neuroprotective effects of BDNF after treatment by
topical application in the conjunctival sac, an experimental model in
which retinal degeneration is induced by light damage was used in animal
models; this model is widely used to study degeneration of the retinal
photoreceptors induced by lengthy exposure to a strong light source (La
Vail et al., 1987; Rex et al., 2003). Photoreceptor death takes place by
apoptosis, and is caused by excessive absorption of photons by the visual
pigment rhodopsin, leading to alteration of the pigment regeneration
cycle which eventually involves the pigmented epithelium cells. The
experimental animal model tested was the albino rat (Surace et al.,
2005), in view of the marked sensitivity of its photoreceptors to light.
The experimental protocol used was modified (Rex et al., 2003) and
expanded from that originally proposed by LaVail's group (LaVail et al.,
1987).

[0103] c) BDNF in an 0.2% solution of sodium carboxymethylcellulose (10
μl of a solution containing 150 μg of BDNF).

[0104] The eyes of rats were treated with the preparations listed above,
and the rats were subjected to light damage. The control eyes were
treated with the carrier solution only. N=4 eyes were treated in each
test group. In particular, after 6 hours' treatment (eye treated with
BDNF and control eye treated with carrier solution only), these rats were
subjected to lengthy exposure to light for 48 hours (animal light-damage
model, light source intensity 1000 1 x). This lengthy exposure to light
induces degeneration of many of the photoreceptors in the retina of
albino rats. The neuroprotection exerted by BDNF was verified by
morphological methods, designed to evaluate the survival of the
photoreceptors, and functional methods, by recording the retinal response
to light (flash electroretinogram [ERG], which is widely used to evaluate
the functional state of the external retina in patients suffering from
retinal disorders). In view of the small number of cones in the rat,
forming the basis of the ERG response under photopic conditions, and the
reduced amplitude of the ERG in photopic conditions in the albino rat,
only the flash ERG was recorded under scotopic adaptation conditions,
expressing the response of the rods of which the rat retina is mainly
composed. The flash ERG (scotopic) was recorded 7 days after the end of
the light-damage period.

[0105] Preparation a)

[0106] FIG. 6 shows the amplitude of the b-wave of the flash ERG according
to luminance under scotopic adaptation conditions. As clearly shown in
the chart in FIG. 6, BDNF in TSP, applied topically, significantly
reduces the effects of light damage on the retinal response to flashes
(flash ERG). In fact, the amplitudes (mean amplitude values expressed in
μV) of the eye treated with BDNF are significantly greater than those
of the control eye, *, p<0.05 (one-way ANOVA).

[0107] Preparation b)

[0108] FIG. 8 shows the amplitudes of the b-wave according to luminance
under scotopic adaptation conditions. The results indicate that BDNF in
saline solution is also able to reduce the alterations of the retinal
response to light induced by light damage (flash ERG). The amplitudes of
the b-wave in the light-damaged eyes of rats treated with BDNF are
greater than those recorded for the control eye; the amplitudes (mean
amplitude values expressed in μV) of the eye treated with BDNF in
saline are significantly greater than those of the control eye, *,
p<0.05 (one-way ANOVA).

[0109] Preparation c)

[0110] FIG. 10 shows the

[0111] amplitude of the b-wave according to luminance under conditions of
adaptation to darkness. The figure demonstrates that the amplitudes (mean
amplitude values expressed in μV) of the eye treated with BDNF in
solution with sodium carboxymethylcellulose are only significantly
greater than those of the control eye, *, p<0.05 (one-way ANOVA) at
the highest luminance values. In conclusion, in terms of functional
recovery of the retinal response to light, sodium carboxymethylcellulose
proved less effective than TSP and saline solution in preventing
impairment of the retinal response to light.

[0112] Subsequently, the effects of topical treatment with the BDNF-based
preparations listed above on degeneration of the retinal photoreceptors
were evaluated in the retinas of the eyes whose flash ERG was recorded.

[0113] The effects of topical treatment with BDNF on photoreceptor
degeneration were quantified by counting the rows of photoreceptors that
survived the light damage and measuring the thickness of the retinal
outer nuclear layer (ONL) which contains the photoreceptor cell bodies.
To perform those measurements, the photoreceptor nuclei were labelled
with propidium iodide.

[0114] Preparation a)

[0115] The results obtained are shown in FIG. 7. FIG. 7A shows the retinal
cross-sections of the eye treated with BDNF (in 0.25% TSP) and the
control eye. To perform those measurements, the photoreceptor nuclei were
labelled with propidium iodide. Regardless of the method used (count of
photoreceptor cell body rows (FIG. 7B) or thickness of the outer nuclear
layer (ONL) (FIG. 7C)), the photoreceptors present in the central and
peripheral retina are significantly (Student's t-test *, p<0.001) more
numerous in the eye treated with BDNF than the eye treated with the
carrier (control).

[0116] It was then demonstrated that BDNF in TSP, when applied topically
in the conjunctival sac, protects the retina from light damage.

[0117] Preparation b)

[0118] FIG. 9 (A) shows the retinal cross-sections of the right eye
treated with BDNF (in saline solution, 0.9% NaCl) and the left (control)
eye treated with saline solution only. The effects of topical treatment
with BDNF on photoreceptor degeneration were quantified by counting the
rows of cell bodies of the photoreceptors that survived the light damage
(FIG. 9B) or measuring the thickness of the retinal outer nuclear layer
(ONL) which contains the photoreceptor cell bodies (FIG. 9C). The
differences between the retinas of the treated eye and the control eye
(count of photoreceptor rows or ONL thickness) proved significant in both
the central and the peripheral retina (Student's t-test *, p<0.001).

[0119] Topical treatment with BDNF in saline increases the number of
photoreceptors that survive light damage in the eye compared with the
control eye.

[0120] Preparation c)

[0121] Finally, the effects of topical treatment with BDNF (in solution
with sodium carboxymethylcellulose) on photoreceptor degeneration were
quantified by measuring the rows of cell bodies of the photoreceptors
that survived the light damage (FIG. 11B) and the thickness of the outer
nuclear retina (ONL) which contains the photoreceptor cell bodies (FIG.
11C).

[0122] Considering the results obtained, in terms of functional recovery
and prevention of photoreceptor degeneration following light damage, it
can be concluded that topical treatment with BDNF in TSP and in saline
solution exercises neuroprotective effects against light damage, whereas
treatment with BDNF in solution with sodium carboxymethylcellulose is
less effective at the same BDNF concentration.

[0123] It was also demonstrated that treatment with BDNF does not induce
functional alterations in the retina, impairing its response to visual
stimuli.

[0124] Glaucoma is a degenerative disorder of the retina which has various
causes, and presents in different forms (it is classified on the basis of
age as congenital glaucoma, infantile glaucoma, juvenile glaucoma or
adult glaucoma; and on the basis of etiopathogenesis as primary glaucoma:
primary open-angle glaucoma or primary angle-closure glaucoma; and
secondary glaucoma induced by other disorders, including iatrogenic
glaucoma). The most common form of glaucoma, namely primary open-angle
glaucoma (POAG), is characterised by increased intraocular pressure which
causes dysfunction and subsequent degeneration of the ganglion cells
associated with atrophy of the optic nerve; the symptoms are gradual loss
of vision, culminating in blindness. The mechanism that causes the
dysfunction and degeneration of the ganglion cells, with atrophy of the
optic nerve, is not yet entirely clear, although the prevalent hypothesis
is that increased intraocular pressure (TOP) induces mechanical damage to
the optic nerve fibres in the lamina cribrosa, and an ischaemic
alteration of the head of the optic nerve and the inner retina. In recent
years, pharmacological treatment has aimed at reducing the IOP, although
a considerable number of patients are resistant to the current
pharmacological treatment and suffer progressive, irreversible loss of
the visual function. There are currently no drugs designed to achieve
neuroprotection of the retinal ganglion cells and the optic nerve fibres
in order to prevent reduction of visual capacity and restore normal
eyesight. In the present patent, we propose the use of topical treatments
with BDNF in the conjunctival sac to increase the retinal BDNF levels in
a stable way so as to counteract the progressive dysfunction of the
ganglion cells, followed by their degeneration and death. This proposal
is partly based on the demonstration that the BDNF receptor, called TrkB,
is expressed in the ganglion cells (Jelsma et al., 1993). To verify our
hypothesis we used the most common experimental model of spontaneous
glaucoma, a double mutant mouse called DBA/2J (John et al., 1998; Chang
et al., 1999). The DBA/2J mouse presents homozygous mutations of two
separate genes; the first is tyrosine-related protein (Tyrp1-/-) coding
for a melanosome protein, and the second is a membrane glycoprotein
(Gpnmb-/-). This mouse is characterised by a progressive increase in
intraocular pressure with progressive loss of the retinal response to
structured visual stimuli, which depends on the inner retina/ganglion
cells; in humans and in the animal model, this retinal response is called
the pattern electroretinogram (P-ERG; Domenici et al., 1991; Ventura and
Porciatti, 2006; Falsini et al., 2008). The dysfunction of the ganglion
cells is followed by a degeneration of said cells with progressive
atrophy of the optic nerve fibres (Ventura et al., 2006). As shown in
FIG. 12, in this murine glaucoma model (DBA/2J), the TOP starts to
increase after 5 months of postnatal life: at 61/2 months the TOP in the
DBA/2J mouse (N=10) already appears significantly higher (t-test;*
p<0.05) than that measured in the normal mouse (C57b1/6J;N=5) and in
the DBA/2J mouse at the age of 5 months (N=9). The chart in FIG. 13 shows
the amplitudes of the response of the inner retina/ganglion cells (P-ERG)
to structured visual stimuli (the visual patterns used as stimulus were
luminance profiles with spatial frequency=0.2 C./deg and 90% contrast),
recorded with corneal electrodes connected to an amplifier and to a
computer for on-line analysis. As shown in FIG. 13, the P-ERG is already
altered in the DBA/2J mouse (CTRL, N=4) at the age of 7 months
(significant reduction in P-ERG amplitudes; Student's t-test, *
p<0.05). From the age of 6.5 months, ie. from the time when the TOP
was stably increased (FIG. 12), a two-week treatment was performed
involving repeated topical applications of BDNF in TSP (one treatment
every 48 hours) in one eye, and the carrier in the other (control eye).
Three different BDNF concentrations were used (N=4 DBA/2J mice per
group): 1, 5 and 15 μg/μl. As shown in the histogram, topical
treatment with BDNF at the concentration of 15 μg/μl (150 μg in
10 μl of solution containing 0.25% TSP, ophthalmic preparation a), but
not at concentrations of 1 and 5 μg/μl, prevented P-ERG alterations
in the DBA/2J mouse (compare the data of the treated eye with the control
eye; Student's t-test, * p<0.05). To establish whether a P-ERG
alteration corresponds to an alteration of the ganglion cells, labelled
with immunohistochemical methods, we used a transcription factor, Brn3b,
expressed in the ganglion cells; mutant mice (Brn3b -/-) for this factor
are associated with an alteration of the ganglion cells (Badea et al.,
2009). FIG. 14 A shows enlargements of retinal preparations in which the
ganglion cells are labelled green with a fluorescent antibody and
analysed by confocal microscopy. The number of labelled ganglion cells is
clearly smaller in the eye of the DBA/2J mouse, in both the central and
the peripheral retina. FIG. 14 B shows the quantitation of the labelled
cells in terms of density (cells/mm2). Two weeks' treatment with
BDNF in TSP at the concentration of 15 μg/μl prevented the
reduction in cells labelled with Brn3b compared with the control eye
treated with the carrier only (Student's t-test, * p<0.05).

[0125] The data reported lead to the conclusion that repeated topical
treatments with BDNF prevent functional alterations of the ganglion cells
and restore the retinal visual capacity in an experimental glaucoma
model. The minimal effective concentration of BDNF able to exert
protective effects on the function of the ganglion cells is 15
μg/μl.